Over nine million platelet units are transfused in the United States every year. The platelets are stored at room temperature to prevent loss of function and thus are particularly susceptible to bacterial contamination since any contaminant bacteria can quickly multiply at room temperature. Platelets are often given to cancer chemotherapy patients to treat platelet depletion and the resulting anemia and risk of bleeding caused by chemotherapy. These patients are also immunocompromised and thus at particular risk from bacterial contamination of the platelets. The number of cases of illnesses and death due to contaminated platelets has only recently been gathered.
Bacterial contamination has been the leading cause of transfusion-related deaths over the past three years (1). Bacterial contamination levels as low as 102 to 103 CFU/ml have been associated with fever and positive blood culture. Studies at Johns Hopkins and Dana-Farber revealed rates of sepsis following platelet transfusion from 0.005% to 0.14%, depending on the site and whether the platelets were derived from random donors or single donor apheresis. In the 33,829 transfusions documented, a total of nine cases of sepsis were found (2, 3). The rate of sepsis appears to be lower than the rate of bacterial contamination in studies of platelet purity. It has been widely suggested that the rate of sepsis from platelet transfusion is underreported for a variety of clinical and regulatory reasons.
Because of the risk of sepsis, the FDA requires platelets to be discarded after five days of storage. For a short period (1984-1986) the FDA permitted platelet storage for seven days, but reversed the regulation after data on bacterial proliferation proved troublesome (4).
The available means for testing for bacteria in platelets are too slow, not sensitive enough, or too cumbersome. One method is culturing the growth of microorganisms from the platelets. The BACT/ALERT system uses this approach. However, this requires culturing for one to three days (5). Another method is gram staining of a sample of platelet concentrate for visual microscopic identification of bacteria. But this requires significant labor and was only sensitive to 106 colony forming units (CFU) per ml (5). Acridine orange staining and fluorescence microscopy improved the sensitivity to 104-105 CFU/ml (5). Visual observation of platelet swirling, or assaying for pH or glucose concentration changes have also been used, but these are not sufficiently sensitive or reliable (5).
A PCR method was used to detect Yersinia enterocolitica. This method was quite sensitive, but took six hours and was specific for only one species (5).
Fluorescent antibodies have also been used to detect bacteria with flow cytometry (5). That has the potential to be sensitive, but is expensive and fairly time consuming, and it only detects the species recognized by the antibodies.
Another method of detecting bacteria involved detection of labeled oligonucleotides that hybridize to bacterial rRNA (6). But the process took four hours.
Bacteria have been detected by luminescence detection of bacterial ATP (7, U.S. Pat. No. 3,933,592). Bacteria are lysed to release their ATP, and the ATP is detected by reaction with luciferase and luciferin to produce light. However, eukaryotic cells have far more ATP than bacterial cells, so even a small contamination with eukaryotic cells gives unreliable results. In one method, blood cells were lysed with TRITON X-100, the debris was separated from bacteria by density gradient centrifugation, and the bacterial cell layer of the gradient was extracted, treated to lyse any bacteria, and assayed for ATP by the luciferin-luciferase assay (8).
New methods to detect bacteria in platelets are needed. Preferably, the methods would be inexpensive, fast, detect bacteria of any clinically significant species, and be sensitive, i.e., detect very low numbers of bacteria. New methods of detecting bacteria in other fluids are also needed. These fluids include whole blood for transfusion, whole blood taken from a patient for diagnosis of sepsis, bone marrow stem cells for a bone marrow transplant, serum, plasma, and urine. Rapid detection of bacteria in urine is needed for diagnostic purposes in both human and veterinary medicine.